Removal of SARS-CoV-2 bioaerosols using ultraviolet air filtration.

Engineering controls play an important role in reducing the spread of severe acute respiratory coronavirus virus 2 (SARS-CoV-2).1 Established technologies such as air filtration, and novel approaches such as ultraviolet (UV)-C light or plasma air ionization, have the potential to support the fight against the coronavirus disease 2019 (COVID-19) pandemic.2 We tested the efficacy of an air purification system (APS) combining UV-C light and high-efficiency particulate air (HEPA) filtration in a controlled environment using SARS-CoV-2 as test organism. The APS successfully removed the virus from the air using UV-C light by itself and in combination with HEPA air filtration.

A Monoclonal Antibody Combination against both Serotypes A and B Botulinum Toxin Prevents Inhalational Botulism in a Guinea Pig Model.

Botulinum neurotoxins (BoNT) are extremely potent and can induce respiratory failure, requiring long-term intensive care to prevent death. Recombinant monoclonal antibodies (mAbs) hold considerable promise as BoNT therapeutics and prophylactics. In contrast, equine antitoxin cannot be used prophylactically and has a short half-life. Two three-mAb combinations are in development that specifically neutralize BoNT serotype A (BoNT/A) and B (BoNT/B). The three-mAb combinations addressing a single serotype provided pre-exposure prophylaxis in the guinea pig inhalation model. A lyophilized co-formulation of six mAbs, designated G03-52-01, that addresses both A and B serotypes is in development. Here, we investigated the efficacy of G03-52-01 to protect guinea pigs against an aerosol exposure challenge of BoNT/A1 or BoNT/B1. Previously, it was found that each antibody demonstrated a dose-dependent exposure and reached maximum circulating concentrations within 48 h after intramuscular (IM) or intravenous (IV) injection. Here we show that G03-52-01, in a single IM injection of G03-52-01 administered 48 h pre-exposure, protected guinea pigs against an aerosol challenge of up to 238 LD50s of BoNT/A1 and 191 LD50s of BoNT/B1. These data suggest that a single IM administration of G03-52-01 provides pre-exposure prophylaxis against botulism from an aerosol exposure of BoNT/A1 or BoNT/B1.

Physiological Responses to Multiple Low-Doses of Bacillus anthracis Spores in the Rabbit Model of Inhalation Anthrax.

Bacillus anthracis spores that are re-aerosolized from surface deposits after initial contamination present significant health risks for personnel involved in decontamination. To model repeated exposure to low dose B. anthracis spores, three groups of seven rabbits were challenged with multiple low-doses of B. anthracis spores 5 days a week for 3 weeks. Mortality, body temperature, heart and respiration rates, hematology, C-reactive protein, bacteremia, and serum protective antigen were monitored for 21 days post-exposure after the last of multiple doses. All rabbits exposed to a mean daily dose of 2.91 × 102 colony forming units (CFU) survived and showed minimal physiological changes attributable to exposure. One of seven rabbits receiving a mean daily dose of 1.22 × 103 CFU died and four of seven receiving a mean daily dose of 1.17 × 104 CFU died. The LD50 was calculated to be 8.1 × 103 CFU of accumulated dose. Rabbits that succumbed to the higher dose exhibited bacteremia and increases above baseline in heart rate, respiration rate, and body temperature. Two rabbits in the mean daily dose group of 1.17 × 104 CFU exhibited clinical signs of inhalation anthrax yet survived. This study provides a description of lethality, pathophysiology, and pathology in a controlled multiple low-dose inhalation exposure study of B. anthracis in the rabbit model. The data suggest that the accumulated dose is important in survival outcome and that a subset of rabbits may show clinical signs of disease but fully recover without therapeutic intervention.

Physiological Responses to a Single Low-Dose of Bacillus anthracis Spores in the Rabbit Model of Inhalational Anthrax.

Credible dose-response relationships are needed to more accurately assess the risk posed by exposure to low-level Bacillus anthracis contamination during or following a release. To begin to fill this knowledge gap, New Zealand White rabbits were implanted with D70-PCT telemetry transmitters and subsequently aerosol challenged with average inhaled doses of 2.86 x 102 to 2.75 x 105 colony forming units (CFU) of B. anthracis spores. Rabbits exposed to a single inhaled dose at or above 2.54 × 104 CFU succumbed with dose-dependent time to death. Death was associated with increases above baseline in heart rate, respiration rate, and body temperature and all rabbits that died exhibited bacteremia at some point prior to death. Rabbits that inhaled doses of 2.06 × 103 CFU or lower survived to the end of the study and showed no or minimal adverse changes in the measured physiological responses in response to the challenge. Moreover, no bacteremia nor toxemia were observed in rabbits that survived to the end of the study. Overall, the data indicate that challenge doses of B. anthracis below the level sufficient to establish systemic infection do not produce observable physiological responses; however, doses that triggered a response resulted in death.

Development of a guinea pig inhalational anthrax model for evaluation of post-exposure prophylaxis efficacy of anthrax vaccines.

A next-generation anthrax vaccine candidate, AV7909, is being developed for post-exposure prophylaxis (PEP) of inhalational anthrax in combination with the recommended course of antimicrobial therapy. Clinical efficacy studies of anthrax countermeasures in humans are not ethical or feasible, therefore, licensure of AV7909 for PEP is being pursued under the US Food and Drug Administration (FDA) Animal Rule, which requires that evidence of effectiveness be demonstrated in an animal model of anthrax, where results of studies in such a model can establish reasonable likelihood of AV7909 to produce clinical benefit in humans. Initial development of a PEP model for inhalational anthrax included evaluation of post-exposure ciprofloxacin pharmacokinetics (PK), tolerability and survival in guinea pigs treated with various ciprofloxacin dosing regimens. Three times per day (TID) intraperitoneal (IP) dosing with 7.5 mg/kg of ciprofloxacin initiated 1 day following inhalational anthrax challenge and continued for 14 days was identified as a well tolerated partially curative ciprofloxacin treatment regimen. The added benefit of AV7909 vaccination was evaluated in guinea pigs given the partially curative ciprofloxacin treatment regimen. Groups of ciprofloxacin-treated guinea pigs were vaccinated. 1 and 8 days post-challenge with serial dilutions of AV7909, a 1:16 dilution of AVA, or normal saline. A group of untreated guinea pigs was included as a positive control to confirm lethal B. anthracis exposure. Post-exposure vaccination with the AV7909 anthrax vaccine candidate administered in combination with the partially curative ciprofloxacin treatment significantly increased survival of guinea pigs compared to ciprofloxacin treatment alone. These results suggest that the developed model can be useful in demonstrating added value of the vaccine for PEP.

Biochemical and aerosol characterization of ricin for use in non-clinical efficacy studies.

Ricin toxin may be used as a biological warfare agent and no medical countermeasures are currently available. Here, a well-characterized lot of ricin was aerosolized to determine the delivered dose for future pre-clinical efficacy studies.  Mouse intraperitoneal (IP) median lethal dose (LD50 ) bioassay measured potency at 5.62 and 7.35 µg/kg on Days 0 and 365, respectively. Additional analyses included total protein, sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, and rabbit reticulocyte lysate activity assay. The nebulizer aerosol produced consistent concentrations (2.5 × 103 , 5.0 × 103 , 1.0 × 104 , and 1.5 × 104  µg/mL) and spray factor values. The aerosol particle size distribution was of sufficient size to deposit in lung alveoli (1.12-1.43 µm). Ricinus communis Agglutinin II (RCA 60), prepared at 19 mg/mL in phosphate-buffered saline, pH 7.8, and stored at -70°C, maintained attributes for toxicity following 1-year storage and aerosolized consistently.

Chikungunya virus infection in Cynomolgus macaques following Intradermal and aerosol exposure.

BACKGROUND: Chikungunya virus (CHIKV) is transmitted via mosquito bite and potentially by aerosol, causing chikungunya fever and arthritic disease in humans. There are currently no licensed vaccines or antiviral therapeutics to protect against CHIKV infection in humans. Animal models recapitulating human disease, especially for transmission by aerosol, are needed for licensure of such medical countermeasures. METHODS: Cynomolgus macaques (CMs) were challenged by intradermal (ID) inoculation or exposure to an aerosol containing CHIKV Ross strain at different target infectious doses (103-107 plaque forming units (PFU)). The clinical and virologic courses of disease were monitored up to 14 days post-exposure. RESULTS: ID infection of CMs led to overt clinical disease, detectable viremia, and increased blood markers of liver damage. Animals challenged by aerosol exhibited viremia and increased liver damage biomarkers with minimal observed clinical disease. All animals survived CHIKV challenge. CONCLUSIONS: We have described CHIKV infection in CMs following ID inoculation and, for the first time, infection by aerosol. Based on limited reported cases in the published literature, the aerosol model recapitulates the virologic findings of human infection via this route. The results of this study provide additional evidence for the potential use of CMs as a model for evaluating medical countermeasures against CHIKV.

Characterization of a Cynomolgus Macaque Model of Pneumonic Plague for Evaluation of Vaccine Efficacy.

The efficacy of a recombinant plague vaccine (rF1V) was evaluated in cynomolgus macaques (CMs) to establish the relationship among vaccine doses, antibody titers, and survival following an aerosol challenge with a lethal dose of Yersinia pestis strain Colorado 92. CMs were vaccinated with a range of rF1V doses on a three-dose schedule (days 0, 56, and 121) to provide a range of survival outcomes. The humoral immune response following vaccination was evaluated with anti-rF1, anti-rV, and anti-rF1V bridge enzyme-linked immunosorbent assays (ELISAs). Animals were challenged via aerosol exposure on day 149. Vaccine doses and antibody responses were each significantly associated with the probability of CM survival (P < 0.0001). Vaccination also decreased signs of pneumonic plague in a dose-dependent manner. There were statistically significant correlations between the vaccine dose and the time to onset of fever (P < 0.0001), the time from onset of fever to death (P < 0.0001), the time to onset of elevated respiratory rate (P = 0.0003), and the time to onset of decreased activity (P = 0.0251) postinfection in animals exhibiting these clinical signs. Delays in the onset of these clinical signs of disease were associated with larger doses of rF1V. Immunization with ≥ 12 µg of rF1V resulted in 100% CM survival. Since both the vaccine dose and anti-rF1V antibody titers correlate with survival, rF1V bridge ELISA titers can be used as a correlate of protection.

Protein- and DNA-based anthrax toxin vaccines confer protection in guinea pigs against inhalational challenge with Bacillus cereus G9241.

In the past decade, several Bacillus cereus strains have been isolated from otherwise healthy individuals who succumbed to bacterial pneumonia presenting symptoms resembling inhalational anthrax. One strain was indistinguishable from B. cereus G9241, previously cultured from an individual who survived a similar pneumonia-like illness and which was shown to possess a complete set of plasmid-borne anthrax toxin-encoding homologs. The finding that B. cereus G9241 pathogenesis in mice is dependent on pagA1-derived protective antigen (PA) synthesis suggests that an anthrax toxin-based vaccine may be effective against this toxin-encoding B. cereus strain. Dunkin Hartley guinea pigs were immunized with protein- and DNA-based anthrax toxin-based vaccines, immune responses were evaluated and survival rates were calculated after lethal aerosol exposure with B. cereus G9241 spores. Each vaccine induced seroconversion with the protein immunization regimen eliciting significantly higher serum levels of antigen-specific antibodies at the prechallenge time-point compared with the DNA-protein prime-boost immunization schedule. Complete protection against lethal challenge was observed in all groups with a detectable prechallenge serum titer of toxin neutralizing antibodies. For the first time, we demonstrated that the efficacy of fully defined anthrax toxin-based vaccines was protective against lethal B. cereus G9241 aerosol challenge in the guinea pig animal model.

Pathology and pathophysiology of inhalational anthrax in a guinea pig model.

Nonhuman primates (NHPs) and rabbits are the animal models most commonly used to evaluate the efficacy of medical countermeasures against anthrax in support of licensure under the FDA's "Animal Rule." However, a need for an alternative animal model may arise in certain cases. The development of such an alternative model requires a thorough understanding of the course and manifestation of experimental anthrax disease induced under controlled conditions in the proposed animal species. The guinea pig, which has been used extensively for anthrax pathogenesis studies and anthrax vaccine potency testing, is a good candidate for such an alternative model. This study was aimed at determining the median lethal dose (LD50) of the Bacillus anthracis Ames strain in guinea pigs and investigating the natural history, pathophysiology, and pathology of inhalational anthrax in this animal model following nose-only aerosol exposure. The inhaled LD50 of aerosolized Ames strain spores in guinea pigs was determined to be 5.0 × 10(4) spores. Aerosol challenge of guinea pigs resulted in inhalational anthrax with death occurring between 46 and 71 h postchallenge. The first clinical signs appeared as early as 36 h postchallenge. Cardiovascular function declined starting at 20 h postexposure. Hematogenous dissemination of bacteria was observed microscopically in multiple organs and tissues as early as 24 h postchallenge. Other histopathologic findings typical of disseminated anthrax included suppurative (heterophilic) inflammation, edema, fibrin, necrosis, and/or hemorrhage in the spleen, lungs, and regional lymph nodes and lymphocyte depletion and/or lymphocytolysis in the spleen and lymph nodes. This study demonstrated that the course of inhalational anthrax disease and the resulting pathology in guinea pigs are similar to those seen in rabbits and NHPs, as well as in humans.

Inhalational monkeypox virus infection in cynomolgus macaques.

An inhalation exposure system was characterized to deliver aerosolized monkeypox virus (MPXV), and a non-human primate (NHP) inhalation monkeypox model was developed in cynomolgus macaques. A head-only aerosol exposure system was characterized, and two sampling methods were evaluated: liquid impingement via an impinger and impaction via a gelatin filter. The aerosol concentrations obtained with the gelatin filter and impinger were virtually identical, indicating that either method is acceptable for sampling aerosols containing MPXV. The mass median aerodynamic diameter (MMAD) for individual aerosol tests in the aerosol system characterization and the NHP study ranged from 1.08 to 1.15 µm, indicating that the aerosol particles were of a sufficient size to reach the alveoli. Six cynomolgus macaques (four male and two female) were used on study. The animals were aerosol exposed with MPXV and received doses between 2.51 × 10(4) to 9.28 × 10(5) plaque forming units (PFUs) inhaled. Four of the six animals died or were euthanized due to their moribund conditions. Both animals that received the lowest exposure doses survived to the end of the observation period. The inhalation LD(50) was determined to be approximately 7.8 × 10(4) pfu inhaled. These data demonstrate that an inhalation MPXV infection model has been developed in the cynomolgus macaque with disease course and lethal dose similar to previously published data.

A three-dose intramuscular injection schedule of anthrax vaccine adsorbed generates sustained humoral and cellular immune responses to protective antigen and provides long-term protection against inhalation anthrax in rhesus macaques.

A 3-dose (0, 1, and 6 months) intramuscular (3-IM) priming series of a human dose (HuAVA) and dilutions of up to 1:10 of anthrax vaccine adsorbed (AVA) provided statistically significant levels of protection (60 to 100%) against inhalation anthrax for up to 4 years in rhesus macaques. Serum anti-protective antigen (anti-PA) IgG and lethal toxin neutralization activity (TNA) were detectable following a single injection of HuAVA or 1:5 AVA or following two injections of diluted vaccine (1:10, 1:20, or 1:40 AVA). Anti-PA and TNA were highly correlated (overall r(2) = 0.89 for log(10)-transformed data). Peak responses were seen at 6.5 months. In general, with the exception of animals receiving 1:40 AVA, serum anti-PA and TNA responses remained significantly above control levels at 28.5 months (the last time point measured for 1:20 AVA), and through 50.5 months for the HuAVA and 1:5 and 1:10 AVA groups (P < 0.05). PA-specific gamma interferon (IFN-γ) and interleukin-4 (IL-4) CD4(+) cell frequencies and T cell stimulation indices were sustained through 50.5 months (the last time point measured). PA-specific memory B cell frequencies were highly variable but, in general, were detectable in peripheral blood mononuclear cells (PBMC) by 2 months, were significantly above control levels by 7 months, and remained detectable in the HuAVA and 1:5 and 1:20 AVA groups through 42 months (the last time point measured). HuAVA and diluted AVA elicited a combined Th1/Th2 response and robust immunological priming, with sustained production of high-avidity PA-specific functional antibody, long-term immune cell competence, and immunological memory (30 months for 1:20 AVA and 52 months for 1:10 AVA). Vaccinated animals surviving inhalation anthrax developed high-magnitude anamnestic anti-PA IgG and TNA responses.

Achieving consistent multiple daily low-dose Bacillus anthracis spore inhalation exposures in the rabbit model.

Repeated low-level exposures to biological agents could occur before or after the remediation of an environmental release. This is especially true for persistent agents such as B. anthracis spores, the causative agent of anthrax. Studies were conducted to examine aerosol methods needed for consistent daily low aerosol concentrations to deliver a low-dose (less than 10(6) colony forming units (CFU) of B. anthracis spores) and included a pilot feasibility characterization study, acute exposure study, and a multiple 15 day exposure study. This manuscript focuses on the state-of-the-science aerosol methodologies used to generate and aerosolize consistent daily low aerosol concentrations and resultant low inhalation doses to rabbits. The pilot feasibility characterization study determined that the aerosol system was consistent and capable of producing very low aerosol concentrations. In the acute, single day exposure experiment, targeted inhaled doses of 1 × 10(2), 1 × 10(3), 1 × 10(4), and 1 × 10(5) CFU were used. In the multiple daily exposure experiment, rabbits were exposed multiple days to targeted inhaled doses of 1 × 10(2), 1 × 10(3), and 1 × 10(4) CFU. In all studies, targeted inhaled doses remained consistent from rabbit-to-rabbit and day-to-day. The aerosol system produced aerosolized spores within the optimal mass median aerodynamic diameter particle size range to reach deep lung alveoli. Consistency of the inhaled dose was aided by monitoring and recording respiratory parameters during the exposure with real-time plethysmography. Overall, the presented results show that the animal aerosol system was stable and highly reproducible between different studies and over multiple exposure days.

Characterization of a therapeutic model of inhalational anthrax using an increase in body temperature in New Zealand white rabbits as a trigger for treatment.

The development of an appropriate animal therapeutic model is essential to assess the potential efficacy of therapeutics for use in the event of a Bacillus anthracis exposure. We conducted a natural history study that showed New Zealand White rabbits exhibited a significant increase in body temperature (SIBT), changes in hematologic parameters, and increases in C-reactive protein and succumbed to disease with an average time to death of approximately 73 h following aerosol challenge with B. anthracis Ames spores. The SIBT was used as a trigger to treat with a fully human monoclonal antibody directed at protective antigen (PA). Ninety percent (9/10) of the treated rabbits survived the lethal inhalational challenge of B. anthracis. Further characterization investigated the protective window of opportunity for anti-PA antibody administration up to 12 h post-onset of SIBT. Eighty-three percent (5/6) of the rabbits treated at SIBT and 100% (6/6) of those treated at 6 h after SIBT survived challenge. Only 67% (4/6) of the rabbits treated at 12 h after SIBT survived. The increase in body temperature corresponded with both bacteremia and antigenemia (PA in the blood), indicating that SIBT is a suitable trigger to initiate treatment in a therapeutic model of inhalational anthrax.

The pathophysiology of inhalational brucellosis in BALB/c mice.

To characterize the clinical presentation and pathophysiology of inhalational brucellosis, Balb/c mice were challenged with Brucella melitensis 16M in a nose-only aerosol exposure chamber. A low dose of 1000 cfu/animal of B. melitensis resulted in 45% of mice with tissue burdens eight weeks post-challenge. The natural history of brucellosis in mice challenged by higher aerosol doses was examined by serial euthanizing mice over an eight week period. Higher challenge doses of 1.00E+05 and 5.00E+05 cfu resulted in positive blood cultures 14 days post-challenge and bacterial burdens were observed in the lung, liver and/or spleens 14 days post-challenge. In addition, the progression of brucellosis was similar between mice challenged by the intranasal and aerosol routes. The results from this study support the use of the Balb/c aerosol nose-only brucellosis mouse model for the evaluation of therapeutics against inhalational brucellosis.

Pathophysiology of the rhesus macaque model for inhalational brucellosis.

The objective of this study was to characterize the rhesus macaque (RM) as a model for inhalational brucellosis in support of the U.S. Food and Drug Administration's (FDA) Animal Rule. The pathophysiology of chronic Brucella melitensis aerosol infection was monitored in two phases that each occurred over an 8-week time period; dose escalation (8 RMs; targeted doses of 5.0E+03, 5.0E+04, or 5.0E+05 CFU/animal or the unchallenged control) and natural history (12 RMs; targeted dose of 2.50E+05 CFU/animal or the unchallenged control). RMs given an aerosol challenge with B. melitensis developed undulating fevers (6/6 phase I; 8/9 phase II), positive enriched blood cultures (5/10; phase II), and bacterial burdens in tissues starting 14 to 21 days postchallenge (6/6 phase I; 10/10 phase II). In addition, 80% (8/10; phase II) of infected RMs seroconverted 14 to 21 days postchallenge. RMs developed elevations in certain liver enzymes and had an increased inflammatory response by 3 weeks postchallenge as shown by increases in C-reactive protein (6/8) and neopterin (4/8), which correlated with the onset of a fever. As early as 14 days postchallenge, positive liver biopsy specimens were detected (2/8), and ultrasound imaging showed the development of splenomegaly. Finally, histopathologic examination found lesions attributed to Brucella infection in the liver, kidney, lung, and/or spleen of all animals. The disease progression observed with the RMs in this study is analogous to human brucellosis pathophysiology. Thus, the results from this study support the use of the RM as an animal model for inhalational brucellosis to evaluate the efficacy of novel vaccines and therapeutics against B. melitensis.

Cynomolgus macaque model for pneumonic plague.

A recombinant vaccine (rF1V) is currently being developed for protection against pneumonic plague. An essential component in evaluating efficacy of the rF1V vaccine is the development of a well-understood animal model that shows similarity to human disease. The objective of this study was to determine the inhaled median lethal dose (LD50), evaluate the pathophysiology of disease and identify appropriate study endpoints in a cynomolgus macaque (CM) model of pneumonic plague. Eighteen CMs were challenged by head-only aerosol exposure with seven dosages of Yersinia pestis CO92. An LD50 of 24 colony forming units was estimated using Probit analysis. Disease pathology was evaluated by blood culture, clinical pathology, histopathology and telemetry. CMs that died became febrile following challenge and died 34-92 h after onset of fever. Bacteremia, increased respiration and heart rate, decreased blood pressure and loss of diurnal rhythm were also observed in conjunction with onset of fever. Histopathological examinations revealed significant findings in the lungs (intra-alveolar neutrophils and fibrinous pleuritis) consistent with pneumonic plague. These data indicate that the disease pathology observed in CMs following aerosol exposure to Y. pestis CO92 is similar to that of pneumonic plague in humans. Thus, the CM is an appropriate model to evaluate efficacy of a recombinant F1V vaccine candidate.

Inhalational botulism in rhesus macaques exposed to botulinum neurotoxin complex serotypes A1 and B1.

A recombinant botulinum vaccine (rBV A/B) is being developed for protection against inhalational intoxication with botulinum neurotoxin (BoNT) complex serotype A, subtype A1 (BoNT/A1), and BoNT serotype B, subtype B1 (BoNT/B1). A critical component for evaluating rBV A/B efficacy will be the use of animal models in which the pathophysiology and dose-response relationships following aerosol exposure to well-characterized BoNT are thoroughly understood and documented. This study was designed to estimate inhaled 50% lethal doses (LD(50)) and to estimate 50% lethal exposure concentrations relative to time (LCt(50)) in rhesus macaques exposed to well-characterized BoNT/A1 and BoNT/B1. During the course of this study, clinical observations, body weights, clinical hematology results, clinical chemistry results, circulating neurotoxin levels, and telemetric parameters were documented to aid in the understanding of disease progression. The inhaled LD(50) and LCt(50) for BoNT/A1 and BoNT/B1 in rhesus macaques were determined using well-characterized challenge material. Clinical observations were consistent with the recognized pattern of botulism disease progression. A dose response was demonstrated with regard to the onset of these clinical signs for both BoNT/A1 and BoNT/B1. Dose-related changes in physiologic parameters measured by telemetry were also observed. In contrast, notable changes in body weight, hematology, and clinical chemistry parameters were not observed. Circulating levels of BoNT/B1 were detected in animals exposed to the highest levels of BoNT/B1; however, BoNT/A1 was not detected in the circulation at any aerosol exposure level. The rhesus macaque aerosol challenge model will be used for future evaluations of rBV A/B efficacy against inhalational BoNT/A1 and BoNT/B1 intoxication.

Antigen-specific Vgamma2Vdelta2 T effector cells confer homeostatic protection against pneumonic plaque lesions.

The possibility that Vgamma2Vdelta2 T effector cells can confer protection against pulmonary infectious diseases has not been tested. We have recently demonstrated that single-dose (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) plus IL-2 treatment can induce prolonged accumulation of Vgamma2Vdelta2 T effector cells in lungs. Here, we show that a delayed HMBPP/IL-2 administration after inhalational Yersinia pestis infection induced marked expansion of Vgamma2Vdelta2 T cells but failed to control extracellular plague bacterial replication/infection. Surprisingly, despite the absence of infection control, expansion of Vgamma2Vdelta2 T cells after HMBPP/IL-2 treatment led to the attenuation of inhalation plague lesions in lungs. Consistently, HMBPP-activated Vgamma2Vdelta2 T cells accumulated and localized in pulmonary interstitials surrounding small blood vessels and airway mucosa in the lung tissues with no or mild plague lesions. These infiltrating Vgamma2Vdelta2 T cells produced FGF-7, a homeostatic mediator against tissue damages. In contrast, control macaques treated with glucose plus IL-2 or glucose alone exhibited severe hemorrhages and necrosis in most lung lobes, with no or very few Vgamma2Vdelta2 T cells detectable in lung tissues. The findings are consist with the paradigm that circulating Vgamma2Vdelta2 T cells can traffic to lungs for homeostatic protection against tissue damages in infection.

Pharmacokinetic considerations and efficacy of levofloxacin in an inhalational anthrax (postexposure) rhesus monkey model.

Because the treatment of inhalational anthrax cannot be studied in human clinical trials, it is necessary to conduct efficacy studies using a rhesus monkey model. However, the half-life of levofloxacin was approximately three times shorter in rhesus monkeys than in humans. Computer simulations to match plasma concentration profile, area under the concentration-time curve (AUC), and time above MIC for a human oral dose of 500 mg levofloxacin once a day identified a dosing regimen in rhesus monkeys that would most closely match human exposure: 15 mg/kg followed by 4 mg/kg administered 12 h later. Approximately 24 h following inhalational exposure to approximately 49 times the 50% lethal doses of Bacillus anthracis (Ames strain), monkeys were treated daily with vehicle, levofloxacin, or ciprofloxacin for 30 days. Ciprofloxacin was administered at 16 mg/kg twice a day. Following the 30-day treatment, monkeys were observed for 70 days. Nine of 10 control monkeys died within 9 days of exposure. No clinical signs were observed in fluoroquinolone-treated monkeys during the 30 treatment days. One monkey died 8 days after levofloxacin treatment, and two monkeys from the ciprofloxacin group died 27 and 36 days posttreatment, respectively. These deaths were probably related to the germination of residual spores. B. anthracis was positively cultured from several tissues from the three fluoroquinolone-treated monkeys that died. MICs of levofloxacin and ciprofloxacin from these cultures were comparable to those from the inoculating strain. These data demonstrate that a humanized dosing regimen of levofloxacin was effective in preventing morbidity and mortality from inhalational anthrax in rhesus monkeys and did not select for resistance.